Multi‐omics technologies and molecular biomarkers in brain tumor‐related epilepsy

Abstract Background Brain tumors are one of the leading causes of epilepsy, and brain tumor‐related epilepsy (BTRE) is recognized as the major cause of intractable epilepsy, resulting in huge treatment cost and burden to patients, their families, and society. Although optimal treatment regimens are available, the majority of patients with BTRE show poor resolution of symptoms. BTRE has a very complex and multifactorial etiology, which includes several influencing factors such as genetic and molecular biomarkers. Advances in multi‐omics technologies have enabled to elucidate the pathophysiological mechanisms and related biomarkers of BTRE. Here, we reviewed multi‐omics technology‐based research studies on BTRE published in the last few decades and discussed the present status, development, opportunities, challenges, and prospects in treating BTRE. Methods First, we provided a general review of epilepsy, BTRE, and multi‐omics techniques. Next, we described the specific multi‐omics (including genomics, transcriptomics, epigenomics, proteomics, and metabolomics) techniques and related molecular biomarkers for BTRE. We then presented the associated pathogenetic mechanisms of BTRE. Finally, we discussed the development and application of novel omics techniques for diagnosing and treating BTRE. Results Genomics studies have shown that the BRAF gene plays a role in BTRE development. Furthermore, the BRAF V600E variant was found to induce epileptogenesis in the neuronal cell lineage and tumorigenesis in the glial cell lineage. Several genomics studies have linked IDH variants with glioma‐related epilepsy, and the overproduction of D2HG is considered to play a role in neuronal excitation that leads to seizure occurrence. The high expression level of Forkhead Box O4 (FOXO4) was associated with a reduced risk of epilepsy occurrence. In transcriptomics studies, VLGR1 was noted as a biomarker of epileptic onset in patients. Several miRNAs such as miR‐128 and miRNA‐196b participate in BTRE development. miR‐128 might be negatively associated with the possibility of tumor‐related epilepsy development. The lncRNA UBE2R2‐AS1 inhibits the growth and invasion of glioma cells and promotes apoptosis. Quantitative proteomics has been used to determine dynamic changes of protein acetylation in epileptic and non‐epileptic gliomas. In another proteomics study, a high expression of AQP‐4 was detected in the brain of GBM patients with seizures. By using quantitative RT‐PCR and immunohistochemistry assay, a study revealed that patients with astrocytomas and oligoastrocytomas showed high BCL2A1 expression and poor seizure control. By performing immunohistochemistry, several studies have reported the relationship between D2HG overproduction and seizure occurrence. Ki‐67 overexpression in WHO grade II gliomas was found to be associated with poor postoperative seizure control. According to metabolomics research, the PI3K/AKT/mTOR pathway is associated with the development of glioma‐related epileptogenesis. Another metabolomics study found that SV2A, P‐gb, and CAD65/67 have the potential to function as biomarkers for BTRE. Conclusions Based on the synthesized information, this review provided new research perspectives and insights into the early diagnosis, etiological factors, and personalized treatment of BTRE.


| INTRODUC TI ON
Epilepsy is a globally prevalent neurological disorder with persistent epileptic seizures, resulting in cognitive, psychological, and social complications. 1,2A systematic review of 222 studies (197 studies on prevalence and 48 studies on incidence) reported that the lifetime prevalence of active epilepsy was 7.60/1000 persons, and the incidence rate was 61.44/100,000 person-years. 3Furthermore, individuals with lower socioeconomic status are more susceptible to developing epilepsy. 4Thus, epilepsy leads to severe consequences in terms of treatment cost and disease burden for both patients and their families, and seizures may result in premature patient death or may significantly affect the patient's quality of life. 5Repeated seizures also affect neurological development in pediatric patients, and some epilepsy patients may experience comorbidities that could further complicate their health condition. 6e following three factors are considered for the early diagnosis of epilepsy: types of seizure, types of epilepsy, and epilepsy syndromes.It is also recommended to identify the underlying causes of epilepsy and its comorbidities (Figure 1). 2,7,8Despite the development of new drugs for treating epilepsy, one-third of patients still experience seizures; therefore, epilepsy remains a worldwide critical public health concern. 9,10ntral nervous system (CNS) cancer is the seventh most common tumor in adults and constitutes 25% of malignant tumors in children; it is also the second leading cause of cancer-related death in young people. 11,12Brain tumors are the leading cause of epilepsy and the second most common histopathological diagnosis in the surgical specimens of patients with epilepsy. 13Brain tumors that originate from the brain tissue are termed primary brain tumors (such as astrocytic tumors, oligodendroglial tumors, and glioneuronal tumors).Malignant brain tumors that metastasize from other parts of the body are termed secondary brain tumors (such as lung cancer and melanoma). 14The incidence of primary brain tumors (including malignant and non-malignant tumors) in the United States was reported as 24.71/100,000 inhabitants per year, 11 which Results: Genomics studies have shown that the BRAF gene plays a role in BTRE development.Furthermore, the BRAF V600E variant was found to induce epileptogenesis in the neuronal cell lineage and tumorigenesis in the glial cell lineage.Several genomics studies have linked IDH variants with glioma-related epilepsy, and the overproduction of D2HG is considered to play a role in neuronal excitation that leads to seizure occurrence.The high expression level of Forkhead Box O4 (FOXO4) was associated with a reduced risk of epilepsy occurrence.In transcriptomics studies, VLGR1 was noted as a biomarker of epileptic onset in patients.Several miRNAs such as miR-128 and miRNA-196b participate in BTRE development.miR-128 might be negatively associated with the possibility of tumor-related epilepsy development.The lncRNA UBE2R2-AS1 inhibits the growth and invasion of glioma cells and promotes apoptosis.Quantitative proteomics has been used to determine dynamic changes of protein acetylation in epileptic and non-epileptic gliomas.In another proteomics study, a high expression of AQP-4 was detected in the brain of GBM patients with seizures.By using quantitative RT-PCR and immunohistochemistry assay, a study revealed that patients with astrocytomas and oligoastrocytomas showed high BCL2A1 expression and poor seizure control.By performing immunohistochemistry, several studies have reported the relationship between D2HG overproduction and seizure occurrence.Ki-67 overexpression in WHO grade II gliomas was found to be associated with poor postoperative seizure control.According to metabolomics research, the PI3K/AKT/mTOR pathway is associated with the development of glioma-related epileptogenesis.Another metabolomics study found that SV2A, P-gb, and CAD65/67 have the potential to function as biomarkers for BTRE. is approximately 1.6% of all cancer cases. 15The incidence of brain metastatic tumors was approximately 25% in individuals with solid tumors. 16Several studies and data sources report varying information on morbidity related to brain tumors; for example, in brain tumor-related epilepsy (BTRE), the morbidity risk largely depends on the types of cancer.7][18][19] Because tumor-related seizures are very persistent, most patients tend to show eventually relapse, even when treated with the best current treatment strategies.The simultaneous treatment of both tumors and epilepsy is more difficult because seizures often signal tumor progression. 18To enable more precise and individualized treatment of brain tumors, the first step is to achieve an accurately diagnosis of the tumors, which requires information on the basic pathological features and molecular biomarkers of these tumors. 19 this article, we reviewed research studies on BTRE published in the last few decades and discussed the present status, development, opportunities, challenges, and prospects in the diagnosis and treatment of BTRE based on omics studies (including genomics, transcriptomics, epigenomics, proteomics, and metabolomics).

| OMI C S S TUD IE S ON THE O CCURREN CE , DE VELOPMENT, AND MECHANIS MS OF DIS E A S E S
Multi-omics analysis includes studies such as genomics, epigenomics, transcriptomics, proteomics, and metabolomics related to an organism or a disease.Omics technology is thought to be highly beneficial for identifying precise tumor markers associated with epilepsy, which can enhance our comprehension of the pathogenetic mechanisms of BTRE. 20The omics biomarkers of BTRE are crucial for the early detection of brain tumors and for selecting appropriate approaches to treat brain tumors and control epilepsy.
During the 1990s and 2000s, the Human Genome Project was conducted, which led to the rapid development of sequencing technologies. 21Simultaneously, rapid advances were achieved in omics technologies and profiles analysis through the multi-omics approach. 19Because BTRE is a complex and multifactorial disease, the evolving omics technologies can enable to analyze the DNA, RNA, metabolites, and proteins in the tumor and peritumoral tissues, and the rapid development of multi-omics technologies can facilitate the investigation of the biological characteristics and molecular biomarkers of BTRE from different perspectives (Figure 2; Table S1).
The application of modern sequencing computational analysis and the integration of multi-omics approaches will further enhance and deepen the understanding of the mechanisms underlying BTRE development; this could contribute to the development of new antiepileptic or antitumor drugs and improve the life quality and prognosis of patients. 22,23

| Genomics technologies and molecular biomarkers
Genomics involves the study of the structure and function of the complete genome of an organism; this includes all regions of the genome, such as coding, noncoding, and regulatory regions of genes. 24though the Sanger dideoxy-nucleotide sequencing method (also termed first-generation sequencing technology) was introduced in 1977, substantial advancement in genomics was achieved in the 2000s through the use of second-generation sequencing technology (also termed next-generation sequencing, NGS). 25 Some approaches known as "third-generation sequencing technologies" have gained popularity in recent years, primarily because they do not require PCR amplification and can simultaneously scan a large number of bases. 26rthermore, the rapid development of other novel technologies such as single-cell sequencing (SNS) exponentially decreased the assay cost. 27Genome-wide association study (GWAS) primarily aims to identifying single-nucleotide polymorphisms (SNPs) in the entire genome and involves the use of microarrays for genotypic analysis of DNA samples from a large case-control cohort. 28Whole-genome F I G U R E 1 International League Against Epilepsy classification and etiologies in epilepsies.variants. 29In contrast, whole-exome sequencing (WES) specifically searches for coding variants in the exon regions and is a cost-effective alternative.WES is the most widely used approach for genomics sequencing, despite concerns regarding the loss of a large number of genes with unknown functions. 30raf murine sarcoma viral oncogene homolog B1 (BRAF) encodes a serine/threonine-specific kinase, which is one of the most important transduction factors of MAPK (RAS-RAF-MEK-ERK) and has the strongest kinase activity in the RAF family.A meta-analysis of 12 studies found that the role of BRAF appears in approximately 35% of BTREs. 31Marucci et al. detected BRAF variants in approximately 50% of tumor cases and demonstrated that these variants were associated with seizures in low-grade tumors. 32Several studies found a statistically significant difference in the age of onset between patients harboring the BRAF V600E mutant and the wildtype gene. 33,34The associated mechanisms reflect tumor cell proliferation regulation through the RAS-RAF-MEK-ERK-MAP kinase signaling pathways, which enhanced tumor cell proliferation. 35rthermore, mice experiments revealed that the BRAF V600E variant induces epileptogenesis in the neuronal cell lineage and tumorigenesis in the glial cell lineage. 36Another study indicated that BRAF variants can activate the mTOR signaling pathway. 37The other tumors developed through alterations in KRAS, RAF, NF1, or FGFR1/2 genes, thus confirming that ganglioma occurs through changes in the RAS-RAF-MAPK pathway. 38e epidermal growth factor receptor (EGFR) gene is associated with seizure risk in glioma patients both preoperatively and postoperatively.patients; by performing immunohistochemical staining, the authors found that patients with lower expression levels of MGMT and EGFR and those with anaplastic oligodendroglioma/anaplastic oligoastrocytoma (AO/AOA) had a higher frequency of postoperative seizure development. 40Isocitrate dehydrogenase (IDH) isoenzyme is one of the most commonly mutated genes in gliomas, and IDH variants occur in over 70% of grade II-III gliomas and a few cases of primary glioblastoma multiforme (GBM). 41Several studies have shown that IDH1 variants result in more easy development of BTRE in preoperative, [42][43][44][45][46] perioperative, 47 and postoperative patients. 48However, some studies have shown that IDH variants are not related to postoperative seizure, 45,49 or are negatively associated with postoperative seizure control. 48A similar result for predicting preoperative seizure was found for IDH2. 50,51IDH1 variants also improve the overall prognosis of glioma patients, which may be useful for the early diagnosis of related seizures. 50,52Furthermore, according to WHO staging, IDH variants are more commonly detected in

Focal
LGGs. 53,54 Several studies have linked IDH variants with glioma-related epilepsy, and the overproduction of D-2-hydroxyglutarate (D2HG) is considered to play a role in neuronal excitation that results in seizure development.The leucine-rich glioma-inactivated gene 1 (LGI1), which encodes a protein rich in leucine and located on human chromosome 10q24, was initially identified as a potential tumor suppressor gene for glioma. 59This gene is downregulated in glioblastoma, thus suggesting that it might have a tumor suppressor effect. 60previous study indicated that the presence of serum LGI1 autoantibodies in some HGG patients may have an epileptic effect. 61ditionally, LGI1 variants have been shown to induce autosomal dominant lateral temporal lobe epilepsy (ADLTE) 62,63 and acquired autoimmune limbic encephalitis (LE), 64,65  RNA-seq offers precise measurements of transcript levels and their isoforms, thereby allowing an impartial and comprehensive mapping of the molecular components, which is known as transcriptome profiling. 71er the last two decades, various occurrences of abnormal gene expression have been documented in different types of epilepsy.Genome Atlas (CGGA) database; 76 the authors found that the high expression level of Forkhead Box O4 (FOXO4) was associated with a reduced risk of epilepsy occurrences.FOXO4 was also found to be a predictor of seizure outcomes in patients with LGGs at 6 months following tumor removal.Fan et al. conducted   an analysis of RNA-seq data from 76 patients with LGGs; 77 the authors found a significant difference in the expression level of RAD50 interactor 1 (RINT1), wherein patients with high RINT1 expression were at a higher risk of developing LGG-related seizures.
The very large G-protein-coupled receptor-1 (VLGR1) protein is the largest cell surface protein currently known and belongs to the Gprotein-coupled receptor family.A study performed RNA-seq of 80 patients with LGGs, and VLGR1 was noted as a biomarker of epileptic onset in patients; furthermore, the authors found that the low expression of VLGR1 was one of the major factors in LGGsassociated seizures and predicted a higher incidence of seizures.
VLGR1 contains multiple Ca 2+ exchanger β (Calxβ) repeats with structures similar to the regulatory domains of Na + /Ca 2+ exchangers; ion channels provide the basis for the regulating excitability in the CNS and are considered to be closely associated with the molecular pathogenesis of epilepsy. 78VLGR1-Ca 2+ binding is localized to the cell surface, and changes in Ca 2+ channels are associated with the molecular pathogenesis of epilepsy.DNA methylation is a chemical modification process that involves the covalent binding of a specific base in the DNA sequence with S-adenosyl methionine as a methyl donor. 91The methods used to detect DNA methylation include methylated DNA immunoprecipitation sequencing (MeDIP-Seq), reduced representation bisulfite sequencing (RRBS), and methylation-sensitive restriction enzyme sequencing (MRE-Seq). 92Currently, the abnormal methylation of gene promoters is used as a molecular marker of tumorigenesis in oncology. 93Various types and sites of histone modifications exist, which can be primarily identified by chromatin immunoprecipitation sequencing (CHIP-Seq). 94

| Proteomics technologies and molecular biomarkers
The traditional methods of protein separation mainly include electrophoresis such as two-dimensional gel electrophoresis (2-DE) and two-dimensional difference gel electrophoresis (2D-DIGE).In recent years, several new techniques have emerged that enable simultaneous separation and identification of proteins, such as gas chromatography (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS) (GC-MS or LC-MS, respectively). 40Xu et al.
performed a quantitative proteomics analysis based on the dynamic changes of protein acetylation in epileptic and non-epileptic gliomas by using label-free MS. 95 The overexpression of the adenosine kinase (ADK) and adenosine deaminase (ADA) genes was found to be associated with neuronal hyperexcitability and seizure activity in temporal lobe epilepsy. 96Huang et al. noted no significant differences in the numbers of ADA-and ADK-positive cells in the tumor tissues between glioma patients with and without seizures. 97This result was consistent with that reported by the European research team of de Groot et al. 98 Aquaporin-4 (AQP-4) is the most abundant AQP (hydrophobic  101 and the authors found that patients with astrocytomas and oligoastrocytomas showed high BCL2A1 expression, which was also associated with poor seizure control.
This finding suggests that BCL2A1 is a potential biomarker in the postoperative seizure control of patients with LGGs.Cx43 is a multifunctional protein that forms hemi-and gap junction channels; it has several binding domains that can interact with specific Cx43-associated proteins and plays a crucial role in many physiological and pathological processes. 102The Cx43 gene is highly expressed in astrocytes under typical physiological conditions.
However, Cx43 expression is reduced as the cell transforms into a malignant one.Sin et al. 103 and Ye et al. 104  epilepsy and the release of D2HG, a chemical structurally related to glutamate, in the TME. 105By performing immunohistochemistry (IHC), several studies have reported the relationship between D2HG overproduction and seizure occurrence. 42,54,55utamate levels were found to be elevated in tumors and adjacent tissues of epilepsy patients with HGGs, and in animal models with xenografted HGG. 56,106The increased expression of the glutamate transporter may play an essential role in the pathogenesis of BTRE. 57Sontheimer et al. found that treatment with sulfasalazine, a system Xc-specific blocker, 107 can reduce the number of spontaneous epileptic events in glioma-implanted mice, thus suggesting that system Xc-mediated glutamate release in the tumor is involved in seizure production.M. S. Sears et al. found that the lack of the system Xc − transporter in mice resulted in decreased epileptogenesis; 108 further studies suggest that this might be partly due to the reduction in the level of AMPA receptor subunit GluA1.The researchers also detected excitatory amino acid transporter 2 (EAAT2) downregulation and system Xc − upregulation, which indicates the key roles of these three biomarkers in BTRE. 57,99Yuen et al. noted that glutamate concentration in glioma and peritumoral tissues was significantly higher in glioma patients with epilepsy than in glioma patients without epilepsy, and glutamate concentration in peritumoral tissues was higher than that in glioma itself; these findings were consistent with those of previous studies. 99Rosati et al. studied samples from 83 newly diagnosed GBM patients by performing IHC; the authors found that glutamine synthetase (GS) expression patterns in neoplastic cells were inversely correlated with the presence of epilepsy, 109 and the GS is an astrocytic enzyme that catalyzes the conversion of glutamate and ammonia to glutamine.
Different from glutamate, γ-aminobutyric acid (GABA) mainly participates in inhibitory signaling pathways.A study indicated that compared to areas without epilepsy surrounding the LGGs, epileptic peritumoral regions contained fewer GABA-containing neurons. 110BAergic networks play an important role in epileptogenesis. 96veral GABA receptor subunits were downregulated in the tissues of patients with gangliogliomas as compared to that in the control group, which could lead to impaired GABAergic tonic inhibition and the subsequent disruption of excitatory-inhibitory connection in the CNS. 111,112The inhibitory effect of GABA is mainly dependent on the K + , Cl − cotransporter (KCC2), which extrudes Cl − from the cell to maintain GABA reversal potential, while the Na + , K + , 2Cl − cotransporter (NKCC1) transports Cl − into the cell to increase the excitatory capability.Elevated NKCC1 expression and decreased KCC2 expression were observed in glioma tumors excised from individuals with medically intractable epilepsy 112,113 ; this finding was further validated by in vitro experiments conducted by Conti et al. 114 Additionally, the alteration in the expression levels of these two transporter proteins in the neocortex around the tumor leads to the excitatory effect of GABA and induces an imbalance between synaptic excitation and inhibition during epilepsy in glioma patients. 114,115n et al. studied five human synapse-associated proteins (SAPs) and found that gamma-aminobutyric acid type A receptor subunit delta (GABRD) 116 was upregulated in the glioma tissue of patients with glioma-associated epilepsy as compared to that in non-epileptic patients; this finding indicated that SAPs may be involved in the pathogenesis of seizures in glioma patients.
The hedgehog-interacting protein (HHIP) gene encodes a membrane glycoprotein and functions as an endogenous antagonist in the hedgehog signaling pathway.Chang et al. studied 135 samples of GBM patients and healthy people by IHC. 117Their results showed that HHIP expression was significantly correlated with seizure.High HHIP expression could be a protective factor, which predicts a low risk for seizure.Previous studies have shown that the inwardly rectifying potassium channel 4.1 (Kir4.1, also known as Kcnj10) plays a key role in potassium hemostasis, which is crucial in maintaining normal neural excitability. 118Zurolo et al. observed the downregulation of Kir4.1 expression in status epilepticus of a rat model of temporal lobe epilepsy 119 ; moreover, the expression level of kir4.1 in the surgical specimens of epileptic patients with a brain tumor was also lower than that in the specimens of non-epileptic patients, which was consistent with the findings of previous studies.The authors also found that the downregulation of Kir4.1 expression corresponded with the prominent upregulation of IL-1β mRNA, thus suggesting the critical role of these biomarkers in tumor-related epilepsy.Ki-67 is an excellent marker for determining the growth fraction of a particular cell population, and its expression level is regularly monitored in many neurosurgery centers to estimate the extent of glioma malignancy and patient prognosis.A lower Ki-67 expression level was detected in patients with preoperative seizures. 39Yang Yuan et al. tested 93 histologically confirmed WHO grade II glioma tissues by using immunohistochemical staining and concluded that Ki-67 overexpression in WHO grade II gliomas is associated with poor postoperative seizure control; thus, Ki-67 is a potential molecular marker to predict poor seizure control after surgery. 120However, a contrasting result was obtained in a later study. 40In the mammalian brain, the N-methyl d-aspartate receptor (NMDAR) is a calcium-permeable subtype of the ionotropic Glu receptor.Gao et al. found that the extrasynaptic NMDA receptor with 2B subunit (NR2B) is substantially phosphorylated at S1013 in neurons present in the peri-glioma area. 121 performing genetic sequencing and an electrophysiology analysis (EEG), Tobochnik et al. found that the presence of H1047R, 122 one of the four commonly observed variants (H1047R, R88Q, E542K, and G118D) in PIK3CA, the PI3K catalytic subunit, was significantly associated with worse seizure control.The synaptic vesicle protein 2A (SV2A) is the binding site for the antiepileptic drug levetiracetam. 123Romoli et al. studied specimens from patients enrolled in the multicenter COMPO study and found the high expression of SV2A can be a protective factor to predict the low risk of BTRE. 124Similar results were obtained by de Groot et al. 125 However, an earlier study by de Groot et al. yielded the opposite result. 126Huang et al. analyzed the clinical data and immunochemistry results of 310 patients with supratentorial astrocytic tumor or oligodendroglial tumor. 127The authors concluded that topoisomerase II (TopoII) positivity is a strong low-risk predictor for preoperative epileptic seizures.
In gliomas, the non-vesicular secretion of glutamate through the cystine-glutamate exchanger (SLC7A11, xCT) is the primary mechanism responsible for the elevated concentration of extracellular glutamate.System XC (SXC), encoded by SLC7A11, is a cystine/glutamate antiporter that releases extracellular glutamate following cystine absorption. 1280][131] Robert et al. observed that in mice with SLC7A11-expressing gliomas, the peritumoral neurons next to the gliomas had depolarized resting potentials and fired more action potentials than the SLC7A11-negative gliomas, thus indicating a hyperexcitable condition in the former. 129Sørensen et al. studied the tumor samples of 229 glioma patients by immunochemistry. 132e authors found that the high xCT expression was significantly associated with seizures at onset, particularly in GBM patients.This finding is consistent with the results of previous studies.were activated and CTNNα was inhibited. 133 summary, proteomics technology is used to identify, quantify, localize, modify, and detect interactions and functions of proteins.
The determination of the special proteins in an organism is essential to accurately and completely discover the in-depth mechanisms of BTRE.

| Metabolomics technologies and molecular biomarkers
Metabolomics/metabonomics is a branch of omics that involves the quantitative analysis of all metabolites present in an organism to investigate their relative relationships during physiological and pathological changes. 134Because of limitations related to detection technology, current metabolomics focuses on metabolites with molecular weights below 1000 Da.Additionally, metabolites are similar across various biological systems; this enables the technologies used in metabolomics research to be more accessible and applicable to different fields. 135Metabolomics can be categorized into two types: nontarget metabolomics and target metabolomics.
Nontarget metabolomics is typically used to broadly screen and identify various metabolites, whereas targeted metabolomics focuses on the analysis of specific metabolites. 136Metabolomics techniques primarily involve the use of nuclear magnetic resonance (NMR), HPLC, GC-MS, and LC-MS.
The thrombospondin (TSP) family is a key regulator of synaptogenesis.Wang et al. analyzed glioma rat models by EEG, and they found that TSP2 overexpression in tumor tissues caused an increase in spine density and excitatory synapses in the peritumoral region, resulting in hyperexcitability in the peritumoral cortical networks. 137The PI3K/AKT/mTOR pathway is associated with the development of glioma-related epileptogenesis. 138A study indicated that PIK3CA may have different effects on peritumoral hyperexcitability depending on the particular variation implicated. 139OLIG2 encodes the oligodendrocyte transcription factor 2 protein, a helix-loop-helix transcription factor that functions as a universal marker of diffuse gliomas.

| A SSOCIATED MECHANIS MS OF EPILEPSY AND B R AIN TUMOR
Epileptogenesis, the process of epilepsy development, is a multifaceted and varied pathophysiological phenomenon.It is triggered by either a genetic variant or an epileptogenic insult, although the cause remains unknown in many patients. 144Currently, the primary causes of epilepsy are identified as structural, genetic, metabolic, and immune factors.However, a considerable proportion of epilepsy cases have an "unknown" cause. 2 It is crucial to understand that the causative factors mentioned above are not solely responsible for inducing spontaneous wiring and disease progression.Before the occurrence of the first spontaneous seizure, several pathological changes occur in the brain tissue (including neurons and glia), such as reactive gliosis and blood-brain barrier compromise.These changes ultimately lead to a state of hyperexcitability, which is associated with a low seizure threshold. 145Although the underlying pathophysiological mechanism is not well understood, seizures may be induced by various factors.glutamatergic signaling pathway.Consequently, seizures occur and contribute to the development of cancer. 129,146Changes in glutamate neurotransmission have been strongly linked to the development in patients with brain tumors, particularly those with highly epileptogenic gliomas.Elevated levels of glutamate have been detected in both tumor and peritumor samples from epilepsy patients with HGG, and in animals implanted with xenografted HGGs. 56,106The increased expression of glutamate transporter may play an essential role in the pathogenesis of tumor-related epilepsy. 57 imbalances in neurotransmitters cause the tumor and the surrounding tissue to become epileptogenic.This implies that the peritumoral tissue is also a part of the "epileptogenic zone." According to some researchers, epileptic activity may originate from areas surrounding the tumor that are within a distance of 1-2 mm from its border, rather than from the tumor itself. 14,147Thus, even a complete resection of a brain tumor does not necessarily mean that the epileptogenic zone has been totally removed; this is because a relevant part of seizures in BTRE can also be generated the surrounding tissue.Part of the dilemma of surgical treatment of tumorinduced epilepsy stems from the following aspect: removal of the tumor alone cannot completely control epilepsy, while excessive removal of the peritumoral tissue can severely impair brain function.
Several preclinical and clinical studies have shown the influence of neurotransmitter imbalance on epileptogenesis in BTRE, in particular, a decrease in inhibitory GABAergic neurotransmission and an increase in excitatory glutamatergic synaptic input. 148Although changes in the extracellular milieu may cause cortical irritability, this depends on the balance between excitatory transmitters, such as glutamate, and inhibitory factors, such as GABA.The inhibitory effect of GABA mainly relies on the KCC2 transporter, which extracts Cl − from the cell to maintain GABA reversal potential.A previous study indicated that compared to areas without epilepsy surrounding an LGG, the epileptic peritumoral regions have fewer GABA-containing neurons. 110Other studies have demonstrated that changes in chloride homeostasis in the peritumoral microenvironment may decrease GABAergic inhibition. 114The disruption of chloride homeostasis is reported in several epilepsy disorders, including BTRE. 115,149KCC2, a cotransporter of potassium and chloride, is specifically mentioned as being involved in this process. 150C2 enables extracellular potassium and chloride release, and is crucial for preserving a chloride gradient across neurons.KCC2 also plays an important role in regulating GABA function by altering intracellular chloride levels to alternate between hyperpolarizing and depolarizing GABAergic signaling.Gliomas show altered KCC2 expression, and the downregulation of KCC2 expression is observed in the peritumoral tissue of mouse glioma models, which corresponds with the occurrence of spontaneous seizures. 150,151C2 is also downregulated in the peritumoral epileptic brain cortex. 114Taken together, these findings suggest that the decreased KCC2 expression could increase intracellular chloride levels, which would then impair inhibitory GABA function, increase excitability, and lower the seizure threshold.Notably, KCC2 may also be downregulated in response to high glutamate concentration, and as such this mechanism may interact with and be compounded by disrupted glutamate homeostasis. 150CC1 is a chloride transporter implicated in epileptogenesis.
NKCC1 is upregulated in human glioblastoma and is responsible for the increase in intracellular chloride levels. 115NKCC1 is also upregulation in ganglioglioma, a tumor invariably linked with seizures, and this coincides with KCC2 downregulation. 112,113The NKCC1 antagonist bumetanide lowers seizure frequency in patients with temporal lobe epilepsy. 152Taken together, these findings indicate that NKCC1 is a viable therapeutic target for BTRE and necessitates more research on its role in the disease. 153Additional molecules involved in ion transport and considered to play a role in epileptogenesis include AQP-4 and Kir4.1.AQP-4 is overexpressed in GBM patients presenting with seizures, while mutations in Kir4.1 are associated with epilepsy in humans and mice models. 99,119,154,155esently, many data retrieval systems (tools and databases) extract a wide range and types of comprehensive data from various resources, including genes, proteins, metabolites, and explore the sequences, structure, function, and classification information.
Databases such as GEO, TCGA, GeneCards, OMIM, ENCODE, Oncomine, and STRIDE are commonly used in research on BTRE (Table 1; Table S1).In-depth mining and improvement of these resources, as well as their effective coupling with omics technology, will be extremely beneficial to study BTRE biomarkers and the mechanisms associated with epilepsy development.

| CON CLUS I ON AND FUTURE PROS PEC T
The generation of BTRE is a complex, multifactor interaction pathophysiology process, involving numerous metabolic changes and pathway changes.In this study, we conducted a systematic review of molecular biomarkers associated with BTRE.We summarized the roles of molecules involved in immune responses, synaptic transmission, and cell cycle control in the development of epilepsy among patients with brain tumors from the perspective of omics (Figure 3).
Compared with a large number of omics studies on tumor tissues, there is a lack of omics studies on peripheral tumor tissues, which is a pity for current research.Epileptic activity is originated from peritumor regions found within 1-2 mm of the tumor border rather than the tumor itself. 156In the current systematic review, only a limited number of studies compared peritumoral tissues with tumor tissues or normal brain cells.Therefore, future studies with larger sample sizes are necessary to investigate the correlation between pathological characteristics of peritumoral tissues and seizures in glioma patients.
According to recent studies, antiepileptic medications like valproic acid and levetiracetam extended the longevity of glioma patients, whereas alkylating treatments like temozolomide reduced the frequency of seizures. 157,158The research on antiepileptic mechanism and antitumor mechanism of many drugs can promote each other, and both can improve our understanding of the mechanisms  In order to more accurately, comprehensively, and systematically understanding the pathophysiological mechanism, omics study in BTRE will be widely performed.Screening the significantly related genes, proteins, metabolites, and metabolic pathways, afterward providing a basis for subsequent functional verification.
Furthermore, clinical application of omics biomarkers is still in the early stages, and there will be more opportunities in the future.

:| 3 of 18 DU
Based on the synthesized information, this review provided new research perspectives and insights into the early diagnosis, etiological factors, and personalized treatment of BTRE.K E Y W O R D S BTRE, genomics, metabolomics, molecular biomarkers, multi-omics, proteomics, transcriptomics et al.
) primarily examines variants in the coding and noncoding regions as well as structural variants regions; however, it has some limitations such as generation of a massive amount of sequencing data, high cost, and unsuitability for identifying low-abundance Yang et al. used the fluorescence in situ hybridization (FISH) technique to analyze DNA obtained from 198 Chinese patients with anaplastic gliomas.Patients with preoperative seizures showed a higher EGFR amplification and a lower Ki-67 expression level. 39Two years later, Yang et al. conducted a similar study that included 147 high-grade gliomas (HGGs)

| 7 of 18 DU
observed a decreased Cx43 expression in the tumor center with the progression of glioma malignancy.Although this evidence indicates that Cx43 could inhibit glioma proliferation, it also implies that the upregulation of Cx43 expression could enhance the invasion and migration of glioma cells; thus, the overall function of Cx43 is complicated.Additionally, Cx43 or GJ/hemi-channels play a role in promoting glioma-associated epileptic and alteration in the tumor microenvironment (TME) by modulating the excitatory neurotransmitter glutamate.102The IDH gene variant induces an abnormal increase of D-2-hydroxyvalerate (D2HG) levels.The mutant IDH does not convert isocitrate to ketoglutarate; instead, it converts ketoglutarate into D2HG.Another study found an association between et al.
tumors trigger seizures.There are several new omics techniques available, but very few have been used to study tumorassociated epilepsy to date.For example, it is well known that tumor is a heterogeneous mass of cells, and slightly different tumor cells may play different roles in the generation of epileptic species.The results obtained by today's common omics techniques are averages of these cells, and the differences have not been studied well.In recent years, the rapid development of single-cell detection technology and spatial omics technology should play an important role in this respect.We expect to see more and more in-depth studies of tumor-associated epilepsy based on these new omics techniques in the future, as the cost decreases the availability of these new technologies.

F I G U R E 3 A
brief framework of the pathogenic mechanisms involved in brain tumor-related epilepsy.
87Previous studies have shown that the lncRNA UBE2R2-AS1 inhibits the growth and invasion of glioma cells and promotes apoptosis through the miR-877-3p/TLR4 pathway.Xu et al. reported that UBE2R2-AS1 expression was positively associ- 881][82][83][84][85]he type of RNAs that do not encode proteins.These RNAs are transcribed from the genome but are not translated into proteins.miRNAsarenoncodingRNAsthatregulategeneexpressionineukaryotes.They are approximately 20-25 nucleotides in length and are produced by cleavage and processing of longer primary transcripts by nuclease.79lncRNAsareRNAmoleculesthatexceed200basepairs in length.Unlike mRNAs, they do not encode proteins but play a crucial role in regulating gene expression at both transcriptional and translational levels through various mechanisms.80miRNAsandlncRNAsarefactorsthatcontrolgeneexpressionandplaya role in neuron development, metabolism, and other activities.[81][82][83][84][85]SeveralmiRNAs,suchasmiR-12886andmiRNA-196b,87participate in BTRE development.Yuan et al. analyzed 53 patients with LGGs and found that miR-128 is negatively associated with the possibility of tumorrelated epilepsy development.86Yangetal.also analyzed the data from The Cancer Genome Atlas (TCGA) by using similar methods and concluded that miR-128 expression was not associated with glioma-associated epilepsy in WHO grade 2 gliomas.88Theavailable evidence, however, cannot confirm or reject the relationship between miR-128 and BTRE.You et al. found higher expression of miR-196b in the tumor tissues of LGG patients with preoperative seizures.
140RTN1 encodes the protein reticulon-1 involved in the neuroendocrine secretion.Lee et al. utilized MRI scanning and RNA-seq approaches to perform gene ex- 143At present, few studies have utilized metabolomics techniques to identify metabolites that are causally related to the BTRE pathogenesis, and this research field is highly worth exploring.
Multi-omics databases and tools commonly used in related studies.Providing functional analysis of proteins by classifying them into families and predicting domains and important sites Proteomics ELM http:// elm.eu.org/ This computational biology resource mainly focuses on annotation and detection of eukaryotic linear motifs (ELMs) by providing both a repository of annotated motif data and an exploratory tool for motif prediction The US data center for the global Protein Data Bank (PDB) archive of 3D structure data for large biological molecules (proteins, DNA, and RNA) essential for research and education in fundamental biology, health, energy, and biotechnology Proteomics Binding DB https:// www.bindi ngdb.org/ bind/ index.jsp Aiming to make experimental data on the noncovalent association of molecules in solution searchable via the WWW.The initial focus is on biomolecular systems, but data on host-guest and supramolecular systems are also important and being included over time Systems and Synthetic Biology is headed by Professor Ivan Mijakovic and is composed of several labs directed by faculty members at the department.The lab and computational facilities are shared among all members of Systems and